The genus Clavariadelphus (Clavariadelphaceae, Gomphales) in China

MycoKeys 70: 89_ | ) (2020) A peer-reviewed open-access journal
doi: 10.3897/mycokeys.70.54 149 RESEARCH ARTICLE Oo Myco Keys

http://mycokeys.pensoft.net Launched to accelerate biodiversity research

The genus Clavariadelphus (Clavariadelphaceae,
Gomphales) in China

Hong-Yan Huang'”, Jie Zhao'", Ping Zhang’, Zai-Wei Ge?, Xian Li', Li-Ping Tang'

| School of Pharmaceutical Sciences and Yunnan Key Laboratory of Pharmacology for Natural Products,
Kunming Medical University, Kunming, 650500, China 2. College of Life Science, Hunan Normal University,
Changsha, 410081, China 3 Key Laboratory for Plant Diversity and Biogeography of East Asia, Kunming
Institute of Botany, Chinese Academy of Sciences, Kunming, 650201, China

Corresponding author: Xian Li (xianlikm@163.com); Li-Ping Tang (lipingtang1 1@qq.com)

Academic editor: Maria P. Martin | Received 11 May 2020 | Accepted 1 July 2020 | Published 29 July 2020

Citation: Huang H-Y, Zhao J, Zhang P, Ge Z-W, Li X, Tang L-P (2020) The genus Clavariadelphus (Clavariadelphaceae,
Gomphales) in China MycoKeys 70: 89-121. https://doi.org/10.3897/mycokeys.70.54149

Abstract

Clavariadelphus species (Clavariadelphaceae, Gomphales) in China were examined using morphology,
molecular phylogenetic analyses of ITS data and chemical reactions. Eleven taxa were identified in China,
including four species known previously to occur in China (C. griseoclavus, C. ligula, C. sachalinensis and
C. yunnanensis), two new record species from China (C. elongatus and C. himalayensis), four novel species
(C. alpinus, C. amplus, C. gansuensis and C. khinganensis) and one species that could not be described due
to the paucity of material. Finally, we also provided a taxonomic key for the identification of Clavariadel-

phus species in China.

Keywords

Clavarioid fungi, taxonomy, molecular systematics, new taxa, species diversity

Introduction

Clavariadelphus Donk (Clavariadelphaceae, Gomphales, Basidiomycota), typified by
C. pistillaris (L.) Donk, is a group of fungi characterised by erect, simple, club-shaped
basidiomes with rhizomorphs at the stipe base, hymenium with (2—) 4-spored basidia,

* Have equally contributed to this work.

Copyright Hong-Yan Huang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC
BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

90 Hong-Yan Huang et al. / MycoKeys 70: 89-121 (2020)

clavate leptocystidia, ellipsoid to amygdaliform, thin-walled, inamyloid basidiospores
and clamp connections at the septa of the hyphae (Methven 1990). The genus is widely
distributed in temperate regions of the Northern Hemisphere and 24 species were
described before this study.

Clavariadelphus has been studied in Europe and North America and important
taxonomic works are available (Corner 1950, 1970; Welden 1966; Smith and Cor-
ner 1967; Petersen 1967, 1972; Smith 1971; Petersen et al. 1974; Methven 1989;
Methven and Guzman 1989). The genus has not received as much attention in Asia,
except for a couple of novel species described from Pakistan (Hanif et al. 2014; Sher
et al. 2018). In China, two novel taxa have been described (Methven 1989; Lu and
Li 2020). To date, only seven Clavariadelphus species have been reported in China,
namely C. griseoclavus L. Fan & L. Xia, C. ligula (Schaeff.) Donk, C. pallido-incarnatus
Methven, C. pistillaris, C. sachalinensis (S. Imai) Corner, C. truncatus Donk and C.
yunnanensis Methven (Methven 1989, 1990; Mao et al. 1993; Yuan and Sun 1995;
Zang 1996; Bau et al. 2003; Mao 2009; Tang and Yang 2014; Tang 2015; Lu and Li
2020). The studies, in which these species were identified, are comparatively brief and
solely based on morphological criteria except C. griseoclavus.

Although Clavariadelphus can be readily distinguished from other members of the
Gomphales, the delimitation of infrageneric taxa is difficult in many cases due to subtle
variations in morphological characteristics and growth habits (Methven 1990). Recently,
molecular techniques have been widely applied and have provided useful information for
species delimitation in systematic fungal studies (Hibbett 2007; Yang 2011). Chemical
reactions are also helpful in delimiting species of many macrofungal groups besides Cla-
variadelphus, including Agaricus, Boletopsis, Chroogomphus, Cortinarius, Hygrophorus, Leu-
coagaricus and Leratiomyces (Corner 1950; Hanif et al. 2014; Siegel and Schwarz 2016).
Scanning electron microscopy (SEM) has been applied to the identification of other mac-
rofungal groups (Zeng et al. 2013; Tang et al. 2014; Huang et al. 2018). However, SEM
of structures of Clavariadelphus has not yet been reported. We mainly examined Chinese
Clavariadelphus collections through analysis of morphological characteristics using light
microscopy and SEM, as well as molecular phylogenetic data, ecological data and chemi-
cal reactions, to better understand species diversity of Clavariadelphus in China.

Materials and methods

Morphological studies

Aside from one collection from the Czech Republic, most specimens of Clavariadelphus
in this study were collected from coniferous forests or mixed coniferous and broad-
leaved forests in North (N) China, Northwest (NW) China and Southwest (SW) China
during the rainy seasons (July-September). Collections and field records are deposited
in the Herbarium of Cryptogams, Kunming Institute of Botany, Chinese Academy of
Sciences (HKAS), Mycological Herbarium, Institute of Mycology, Chinese Academy of
Sciences (HMAS), Mycological Herbarium of Hunan Normal University (MHHNU)

Clavariadelphus species al

and Mycological Herbarium of Pharmacy College, Kunming Medical University
(MHKMU) (Appendix 1). Specimens and their habitats were photographed in situ.
Relevant metadata, such as altitude, latitude, longitude and nearby tree associates were
recorded in the field. Detailed notes on macro-morphological descriptions were taken
from fresh material and colour codes were from Kornerup and Wanscher (1981).

Light microscopy

Micro-morphological characteristics were observed under a light microscope (Leica
DM 2500). Preparations were made from dried specimens. Tissue fragments of dried
materials were sectioned, mounted in 10% KOH and observed. The abbreviation
[n/m/p] means n basidiospores measured from m basidiomes of p collections. Dimen-
sions for basidiospores are given as (a) b—c (d). The range of b—c contains a minimum
of 90% of the measured values. Extreme values, i.e. a and d are given in parentheses.
Q is used to denote the length/width ratio of basidiospores in the side view, whereas
Q refers to the average Q value of all basidiospores + standard deviation.

Scanning electron microscopy

The material was sampled and directly used from herbarium collections. The hyme-
nium and basal mycelium from dried specimens were mounted on to aluminium stubs
coated with gold palladium. Basidiospores and hyphae of the basal mycelium were
observed and micrographs were taken with a ZEISS Sigma 300 scanning electron mi-
croscope at 7.0 kV accelerating voltage.

Chemical reactions

Seven chemical reagents were used: 10% (w/v) KOH, 10% (w/v) FeCl, 10% (w/v)
FeSO,, 10% NH,OH, 10% (w/v) phenol, Melzer’s reagent and 95% (v/v) ethanol.
Small slices of tissue were taken from the hymenium of the basidiomes. ‘The reagents
were systematically added to the depression in plates so that each piece of tissue was
submerged in several drops of a single reagent. Positive colour reactions were recorded
immediately following the application of reagents.

DNA extraction, PCR and DNA sequencing

Total genomic DNA was isolated from dried materials using a modified CTAB method
(Doyle 1987) with a prolongation of the extraction period as necessary. For PCR reactions,
the nuclear ribosomal DNA internal transcribed spacer (ITS) region was amplified using
primers ITS5 and ITS4 (White et al. 1990). The PCR amplification mix consisted of a

92 Hong-Yan Huang et al. / MycoKeys 70: 89-121 (2020)

total volume of 25 ul containing 2.5 pl of 10 x amplification buffer (with MgCl), 0.5 pl
dNTP (200 pM), 0.2 pl Taq DNA polymerase (5 U/ul), 1 pl of each primer (10 uM), 1 pl
DNA template and 18.8 l sterile water. PCR reactions were performed with an initial
denaturation at 94 °C for 4 min; 38 cycles of denaturation at 94 °C for 40 s, annealing at
54 °C for 40 s, extension at 72 °C for 60 s; and a final extension at 72 °C for 8 min. PCR
products were checked on 1% agarose gel. Successful reactions were sequenced using an
ABI 3730 DNA Analyzer (Sangon, Shanghai, China) with both PCR primers. The DNA
sequences were used as queries in NCBI BLAST searches to rule out contamination. The
forward and reverse sequences were assembled with SeqMan (DNASTAR Lasergene 9)
and their quality controlled according to the guidelines of Nilsson et al. (2012). Novel and
already available sequences were aligned by using MAFFT version 7 (Katoh and Standley
2013). The alignment was manually adjusted in BioEdit version 7.0.9 (Hall 1999) and
trimmed in trimAl version 1.2 (Capella-Gutiérrez et al. 2009).

Phylogenetic analyses

Two phylogenetic tree inference methods, Randomised Accelerated Maximum Likeli-
hood (RAxML) and Bayesian Analysis (BA), were used to analyse the ITS sequence data.
The programme RAxML version 7.0.3 (Stamatakis et al. 2008) was used to infer a maxi-
mum likelihood tree with bootstrap support values and the GTRGAMMA was selected
as a default model. The programme MrBayes version 3.2.6 (Ronquist et al. 2012) was
run using a Markov Chain Monte Carlo (MCMC) tree sampling procedure. The ITS1,
5.88 and ITS2 loci were extracted from the aligned ITS dataset, allowing the selection
of substitution models for each partition. Aligned sequences were partitioned into ITS1
(1-270), 5.8S (271-429) and ITS2 (430-703). Nucleotide substitution models based on
the Akaike Information Criteria (AIC) data were obtained in PartitionFinder 2 (Lanfear
et al. 2016). The selected models were GITR+G for ITS1, K80 for 5.8S and HKY+G for
ITS2. After four simultaneous Markov chains running with 7,000,000 generations and
sampling every 100 generations, the average deviation of split frequencies was 0.004022
at the end of the run. Burn-in values were determined in Tracer v1.7 (Rambaut et al.
2018). Effective sample sizes were well over 200 for all sampled parameters for each run
and the initial 20% of the samples was discarded. Bayesian Posterior Probabilities (PP)
were calculated for a majority consensus tree of the retained Bayesian trees.

Results

Taxonomic identification based on morphological data

Fifty specimens of Clavariadelphus were examined in this study. Six species were pre-
viously reported from China, except the late described one, C. griseoclavus. However,

Clavariadelphus species 23

the re-examination of available vouchers confirmed the occurrence of only three of
these species, specifically C. ligula, C. sachalinensis and C. yunnanensis. Our motr-
phological observations revealed that nine taxa, including three species previously
identified in China (C. ligula, C. sachalinensis and C. yunnanensis), two species that
have not been previously reported from China (C. elongatus and C. himalayensis) and
four novel species (C. alpinus, C. amplus, C. gansuensis and C. khinganensis), were
identified on the basis of morphological characters. So far, there are ten described
taxa in China, including C. griseoclavus which is recently published.

Taxonomic identification based on molecular data

The ITS dataset comprised 27 ingroup taxa including the type species C. pistillaris
and three outgroup taxa, with 64 sequences in total. The length of the alignment was
703 aligned bases (TreeBASE accession 24163). Three species of Lentaria Corner
and Kavinia Pilat were chosen as outgroups in the dataset, based on a previous study
(Giachini et al. 2010).

In the phylogeny, based on ITS sequences, few differences in the topology of
major clades were detected between the ML and Bayesian analyses. Twenty-seven
phylogenetic species were recovered, amongst which, eleven species were from Chi-
na, including one with a GenBank accession JQ991679 from Zhejiang Province,
China, which might represent a separate species in the tree (Fig. 1). Clavariadelphus
sachalinensis formed a distinct lineage with high support and was sister to the rest of
the genus. Seven Chinese lineages, namely C. amplus, C. elongatus, C. griseoclavus,
C. himalayensis, C. ligula, C. khinganensis and C. yunnanensis, were strongly support-
ed as monophyletic groups. The other two species from China, namely C. alpinus
and C. gansuensis, were each represented by only one specimen in the phylogenetic
tree. The sister of each Chinese taxon is discussed below.

Taxonomic identification based on chemical reactions

Steglich et al. (1984) proposed that a positive ferric salts reaction of the basidiomes was
indicative of the presence of pistillarin in the basidiomes of Clavariadelphus. To a large ex-
tent, Methven’s study (1990) supported this hypothesis, excluding one exception (C. cokeri
V.L. Wells & Kempton). Methven (1990) mentioned the negative ferric salts reaction of
some species might be the result of pistillarin being present in too low concentrations or
the result of samples affected by pesticides during storage. In our study, most species have
positive reactions with four reagents (FeCl, KOH, NH,OH and phenol), but all species
from China showed a negative reaction to FeSO bs Melzer’s reagent and ethanol. The results
of the chemical testing in this study are summarised in Table 1. As those specimens are
preserved, pesticides are used regularly. Thus, we agree with Methven’s argument (1990).

94 Hong-Yan Huang et al. / MycoKeys 70: 89-121 (2020)

Clavariadelphus sachlinensis EU624410 USA
C. sachlinensis EU624411 USA
4100/1 | C. sachlinensis EU624408 USA
C. sachlinensis EU834196 USA
C. sachlinensis MHHNU 7816 Inner Mongolia China

°C. truncatus HQ650728 Canada
C. gansuensis HKAS 76487 Gansu China
C. pakistanicus HQ379937 Pakistan

"C. pakistanicus" MT012805 India

C. amplus HKAS 54876 Yunnan China
Wc. ampius HKAS 49229 Sichuan China
C. ampilus HKAS 76577 Gansu China

C. amplus HMAS 250466 Yunnan China

C. amplus HKAS 53797 Sichuan China
mplus HMAS 132008 Qinghai China
Clavariadelphus sp. MH304404 Mexico
Clavanadelphus sp. KT874982 Mexico

100/1

Ca
100/1
51/- Fs
"C. truncatus” AJ292288 Spain

C. alpinus HKAS 57396 Yunnan China
97/0.92 C. truncatus MT452508 Italy
"C. occidentalis" EU669202 USA
96/0.98, C. khinganensis MHHNU 7789 Inner Mongolia China
C. khinganensis MHKMU H.Y. Huang 368 Jilin China
*C. truncatus” DQ097871 Canada
1000.95, Ciavariadelphus sp. MKO28378 Switzerland
Sie C. pistillaris MT452507 Italy

“C. occidentalis" KU973835 Tunisia
C. elongatus HKAS 76589 Gansu China
C. elongatus HKAS 50742 Sichuan China
C. elongatus MG768848 Pakistan
C. elongatus HKAS 50801 Sichuan China
C. elongalus MG768847 Pakistan
C. elongatus HMAS 260746 Yunnan China
C. yunnanensis HKAS 57731 Yunnan,China

C. yunnanensis HKAS 77288 Yunnan China
C. yunnanensis HKAS $7659 Yunnan China
C. yunnanensis HMAS 250510 Yunnan China
C. yunnanensis HKAS 58789 Yunnan China
C. yunnanensis HKAS 62644 Yunnan China
C. yunnanensis HKAS 54849 Yunnan China

C. yunnanensis HKAS 63558 Yunnan China
Clavariadelphus sp. DQ974709 USA
92/0.98, C. americanus MKS75228 USA
100/1 ) ©. ligula HMAS 51688 heilongjiang China
C. ligula HMAS 41146 Czechoslovakia
¢. ligula HMAS 35954 heilongjiang China
100/1 | ©. himalayensis HKAS 58811 Yunnan China
C. himalayensis HKAS 50684 Yunnan China
"C, pistillaris” MH979250 USA
Clavariadelphus sp. JQ991679 Zhejiang China

-1- ©. mucronatus EU526000 USA
C. subfastigiatus JX275756 USA
C. subfastigiatus EU669207 USA
C. unicolor MN906166 USA

56-
96/1

So/-

1400/1

100/1 | C. griseaclavus MT302370 Shanxi China

' C. griseoclavus MT302371 Shanxi China
100/1 | ¢. occidentalis EU669203 USA
C. occidentalis EU834195 USA

Lentania byssiseda FJ596788 USA

100/1 Kavinia alboviridis KJ140634 USA
Brg Tk. himantia K140598 USA

Figure |. Phylogenetic tree of Clavariadelphus based on ITS sequence data. RAxML BP values (= 50%)
are shown above branches, Bayesian posterior probabilities (= 0.90) are shown above branches; new taxa

are marked in red.

Taxonomy

1. Clavariadelphus alpinus J. Zhao & L.P. Tang, sp. nov.
MycoBank No: 830258
Figs 2a, 3a, 4a, 5a, 6a, b

Diagnosis. This species is distinguished from other taxa in Clavariadelphus by the
light yellow, clavate basidiomes with enlarged apex, broadly ellipsoid basidiospores,
hyphae of the basal mycelium with nipple-shaped protuberances and basidiomes turn-
ing lemon-chiffon in KOH.

Etymology. Latin “alpinus” refers to this species occurring in high-altitude areas.

Description. Basidiomes up to 12 cm high, 0.9 cm diam. at the base, enlarged up-
wards to 2 cm diam., simple, initially cylindrical to subcylindrical, then narrowly clavate to
clavate, laterally compressed in age; /ymenium initially smooth, then longitudinally rugose,
light yellow (4A4—5) to yellow or yellowish-orange, apricot-yellow, light orange-yellow
(4A6-7) or (SA5-6); apex subacute to obtuse, smooth to rugose, concolorous with the hy-
menium; surface not staining when cut or bruised; base terete, smooth, white to cream; my-
celial hyphae white; flesh initially solid, then soft and spongy upwards as the apex enlarges,

white not staining on exposure. Odour and taste not recorded. Spore deposit not recorded.

Clavariadelphus species wie)

Table |. Chemical reactions of representative species of Clavariadelphus from China.

Taxa KOH FeCl, NH,OH Phenol Ethanol Melzer’s FeSO,

reagent
C. alpinus 3B8 - 6A8 - — — -
C. amplus 12A4 1A8 2A8 2A5 - - —
C. elongatus 2A5 1A8 6A8 _ = — S
C. gansuensis 9B7 1A8 2A8 2A8 - — —
C. himalayensis 5B7 30A8 6A8 — — - -
C. khinganensis 2A5 = - — — - _
C. ligula 3B8 - 6A8 - — - -
C. sachalinensis 2A5 30A8 6A8 _ — _ _
C. yunnanensis 5B7 30A8 2A8 2A5 7 - -
Note: “—” indicates negative reactions.

Hymenium extending over the apex of basidiomata, composed of basidia and lep-
tocystidia. Basidia 65-85 x 8-10 um, clavate, hyaline, thin-walled, (2—, 3—) 4-spored,
sterigmata 8-12 um in length. Basidiospores [20/1/1] (7.4—) 7.8-9.6 (-10.1) x 5.5
(—5.1)-7.4 um, Q = 1.25-1.55 (1.58), Q_ = 1.38 + 0.10, broadly ellipsoid, ovate
or amygdaliform, with a small apiculus, inamyloid, thin-walled, hyaline in KOH,
smooth. Leptocystidia 45-55 x 2.8—4.2 um, scattered amongst and scarcely projecting
beyond the basidia, cylindrical to narrowly clavate, thin-walled, smooth, hyaline, non-
pigmented, clamped, inflated apically at maturity and at times, with apical or subapical
branches. Mycelial hyphae 2-4 um diam., interwoven or aggregated into rhizomorphic
strands, branched, clamped; hyphal walls echinulate with light microscopy, covered
with massive nipple-shaped protuberances without crystals with SEM.

Chemical reactions (dried basidiomes). KOH = positive, lemon-chiffon; NH,OH
= positive, orange; ethanol, FeCl,, FeSO,, Melzer’s reagent and phenol = negative.

Known distribution and ecology. SW China, Yunnan Province. Solitary on the
ground in forests dominated by conifers (e.g. Abies georgei) at elevations of approxi-
mately 3700 m.

Materials examined. Cutna. Yunnan Province: Shangri-la Prefecture, Bita Lake,
24 August 2009, approximately 3700 m elev., B. Feng 667 (HKAS 57396, Holotype).

Comments. Clavariadelphus alpinus is well characterised by its yellow basidiomes,
broadly ellipsoid basidiospores, hyphae of the basal mycelium with nipple-shaped pro-
tuberances, the apex of the basidiomes having a positive reaction to NH,OH and
KOH and distribution at high elevations in SW China in association with conifers.

Morphologically, this taxon is similar to C. khinganensis. However, C. khinganensis
has light brown-tan basidiomes, more elongated basidiospores (Q = 1.6—2.2), negative
reaction to NH,OH and distribution at lower elevations in NE China.

In the ITS phylogeny, this species is a sister species of C. truncatus with strong sup-
port (Fig. 1). However, C. truncatus differs from C. alpinus by having dark coloured
basidiomes from yellow to cinnamon-brown or brown, broader apices (up to 3.5 cm)

and larger basidiospores (10.3—12.6 x 5.5—7.1 um from neotype; Methven 1990).

Hong-Yan Huang et al. / MycoKeys 70: 89-121 (2020)

Figure 2. Clavariadelphus species in China. a C. alpinus (HKAS 57396, holotype) b, ¢ C. amplus (HKAS
54876, holotype) d, e C. elongatus (d from HKAS 50742 e from HKAS 76589) f C. gansuensis (HKAS
76487, holotype) g C. himalayensis (HKAS 58811) h,i C. khinganensis (h from MHHNU 7789, holo-
type i from MHKMU HY. Huang 368) j C. sachalinensis (MHHNU 7816) k, I C. yunnanensis (k from
HKAS 49398 I from HKAS 58789).

Clavariadelphus species 97

000 0005 G°C
d00 Jpop YE

00,00 000
OOUD

Figure 3. Basidiospores of Clavariadelphus under light microscope. a C. alpinus (HKAS 57396, holo-
type) b C. amplus (HKAS 54876, holotype) ¢ C. elongatus (HKAS 76589) d C. gansuensis (HKAS 76487,
holotype) e C. himalayensis (HKAS 58811) f C. khinganensis (MHHNU 77839, holotype) g C. ligula
(HKAS 35954) h C. sachalinensis (MHHNU 7816) i C. yunnanensis (HKAS 57659).

2. Clavariadelphus amplus J. Zhao, L.P. Tang & Z.W. Ge, sp. nov.
MycoBank No: 830271
Figs 2b, c, 3b, 4b, 5b, 7a, b.

Diagnosis. ‘This species is unique in its pink-orange basidiomes with enlarged, trun-
cate and sterile apices, ellipsoid basidiospores, hyphae of the basal mycelium with nip-
ple-shaped protuberances and prism-like crystals and basidiomes turning cherry-red in
KOH. It differs from C. truncatus by the latter’s darker coloured basidiomes, narrower
apices and larger basidiospores.

Etymology. Latin “amplus” refers to the enlargement of the apex of the basidiomes.

Description. Basidiomes up to 15 cm high, 0.5—1 cm diam. at the base, enlarged
upwards to 3—7.5 cm diam. near apex; /ymenium initially smooth, longitudinally ru-
gulose in age, pruinose, pinkish-orange (7A5—7), paler downwards, greyish-orange
(5B4—5); apex initially obtuse or broadly rounded, finally truncate, depressed, surface
rugose to rugulose, more or less darker than the hymenium, apricot-yellow (5B6—7)
to pink-orange, reddish-orange (7A7—8) or red-orange (7B7-—8) at maturity; surface
slowly staining light brown or light leather-brown (7D6-—7) to brown (7E6—7) when

cut or bruised, staining more conspicuously downwards; base simple, terete, nearly

Hong-Yan Huang et al. / MycoKeys 70: 89-121 (2020)

Figure 4. Basidiospores of Clavariadelphus under SEM. a C. alpinus (HKAS 57396, holotype)
b C. amplus (HKAS 54876, holotype) ¢ C. elongatus (HKAS 76589) d C. gansuensis ( HKAS 76487,
holotype); e, f C. himalayensis (HKAS 58811) g C. khinganensis (MHHNU 7789, holotype) h C. yun-
nanensis (HKAS 57659).

Clavariadelphus species

Figure 5. Hyphae of basal mycelium from Clavariadelphus under SEM. a C. alpinus (HKAS 57396,
holotype) b C. amplus (HKAS 54876, holotype) ¢ C. elongatus (HKAS 76589) d C. gansuensis (HKAS
76487, holotype) e C. himalayensis (HKAS 58811) f, g C. sachalinensis (f from HKAS 33844; g from
MHHNU 7816) h C. yunnanensis (HKAS 57659).

100 Hong-Yan Huang et al. / MycoKeys 70: 89-121 (2020)

a
b

Figure 6. Microscopic features of Clavariadelphus alpinus (HKAS 57396, holotype). a Basidia b Leptocystidia.

10 um

Figure 7. Microscopic features of Clavariadelphus amplus (HKAS 54876, holotype). a Leptocystidia and

immature basidia b Basidia.

Clavariadelphus species 101

smooth, cylindrical to subcylindrical, pruinose; mycelial hyphae interwoven, white;
flesh solid initially, then soft and spongy upwards as the apex enlarges, white, slowly
staining light leather-brown (7D6—7) to brown (7E6—7) on exposure. Odour pleasant.
Taste not distinctive. Spore deposit not recorded.

Hymenium limited to the sides of basidiomes, composed of basidia and leptocyst-
idia; the apex of basidiomata is composed of sterile elements 18-28 x 5—8 um, clavate,
thin-walled, smooth, clamped. Basidia 85-95 x 8-12 um, clavate, hyaline, thin-
walled, (2—) 4-spored, sterigmata 9-11 pm in length. Basidiospores [40/2/2] 8.2-11.0
x 5.1-6.4 um, Q = (1.36-) 1.38-2.00 (-2.18), Qa 17> £:0:17, ellipsoid to broadly
ellipsoid, ovate or amygdaliform, with a small apiculus, inamyloid, thin-walled, hy-
aline in KOH, smooth. Leptocystidia 45-70 x 2.8-3.8 um, scattered amongst and
scarcely projecting beyond the basidia, cylindrical to narrowly clavate, thin-walled,
smooth, hyaline, non-pigmented, clamped, inflated apically at maturity and at times,
with apical or subapical branches. Mycelial hyphae 2-4 um diam.., parallel, interwoven
or aggregated into rhizomorphic strands, branched, clamped; walls thin or irregularly
slightly thickened, the hyphal walls echinulate with light microscopy, covered with
nipple-shaped protuberances, as well as encrusted with prism-like crystals (up to 6 um
long) that are insoluble in KOH.

Chemical reactions. (dried basidiomes): FeCl, = positive, green-yellow; KOH =
positive, cherry-red or pink; NH,OH = positive, golden-rod or vivid yellow; phenol =
positive, light yellow; ethanol, FeSO . and Melzer’s reagent = negative.

Known distribution and ecology. NW China and SW China, and India. Gre-
garious habit on the ground in conifer or mixed conifer forests (e.g. Abies spp. and
Picea spp.) at elevations ranging from 3000-3950 m.

Materials examined. Cuina. Gansu Province: Zhouqu Prefecture, under Abies
spp., 6 August 2005, X. 77 Zhu 728 (HKAS 76577). Qinghai Province: Qilian moun-
tains, 38°6.00'N, 100°7.03’E, alt. 3000 m, 21 August 2004, H.A Wen 4305 (HMAS
132008); same location and date, Q.B. Wang 438 (HMAS 97090). Sichuan Province:
Seda Prefecture, Picea-Juniperus forests, 31°43.20'N, 100°43.17'E, alt. 3775-3925 m,
6 August 2005, Z.W. Ge 783 (HKAS 49278); Litang Prefecture, 5 August 2007, Z.W
Ge 1712 (HKAS 53797). Tibet: Linzhi City, 29°20.07'N, 094°18.00'E, alt. 3850 m,
19 July 2004, YH. Wang 125 (HMAS 97248); Jilong Prefecture, on the ground in
coniferous woods, 12 September 1990, /. ¥ Zhuang 3814 (HMAS 59867); Chengdu
City, under forests dominated by Picea spp., 31°30.43'N, 097°20.07'E, alt. 3480—
3550 m, 17 August 2004, ZW. Ge 381 (HKAS 46160); Riwoge Prefecture, under
Picea spp., 31°14.27'N, 096°31.92'E, alt. 3890 m, 12 August 2004, Z.W Ge 340
(HKAS 46120). Yunnan Province: Shangri-La Prefecture, Haba Snow Mountains,
alt. 2800 m, 15 August 2008, L.P Tang 645 (HKAS 54876, Holotype); Shangri-La
Prefecture, 27°28.13'N, 099°25.03'E, alt. 3600 m, 15 August 2008, 772. Wei 172
(HMAS 250466).

Comments. Clavariadelphus amplus is distinctive by its pink-orange to red-orange,
bright basidiomes, obviously enlarged, truncate, depressed, sterile apices (up to 7.5 m
diam.) at maturity, large basidiospores (8.2—11.0 x 5.1-6.4 um), gregarious habit at
high elevations, base mycelial hyphae with nipple-shaped protuberances and prism-

102 Hong-Yan Huang et al. / MycoKeys 70: 89-121 (2020)

like crystals and a cherry-red staining reaction to KOH. It is sold as an edible mush-
room in markets in SW China. This taxon has a wide distribution in NW and SW
China, including Gansu, Qinghai, Sichuan, Tibet and Yunnan Provinces. The data
from GenBank (accession MT012805) also indicated its distribution of India.

This species was previously referred to as either C. pallido-incarnatus (Yuan and
Sun 1995) or C. truncatus (Mao et al., 1993; Zang 1996; Mao 2009; Tang and Yang
2014; ‘Tang 2015). Clavariadelphus pallido-incarnatus, a species described from the Pa-
cific Northwest in North America, has pale pinkish-cinnamon basidiomes with fertile,
non-truncated apices, no reactivity to KOH and habitat preference for coastal forests of
Sequoia sempervirens and Picea sitchensis (Methven 1990). Clavariadelphus truncatus from
Europe is readily confused with C. amplus as they have similar size and truncate sterile
apex. However, C. truncatus has dark coloured basidiomes from yellow to cinnamon-
brown or brown, narrower apices (up to 3.5 cm) and larger basidiospores (10.3-12.6 x
5.5-7.1 um from neotype; Methven 1990). Clavariadelphus unicolor (Berk. & Ravenel)
Corner, is also from North America and has enlarged sterile apices, but it is distinct in its
reddish-brown to violet-brown basidiomes, narrow basidiospores with Q_ 2.1, a golden-
yellow reaction to KOH and association with deciduous trees (Methven 1990).

So far, there are two species with sterile apices found in China, C. amplus and C.
gansuensis. However, C. gansuensis has a narrower apex (up 1.6 cm), slightly broader
basidiospores with a lower Q value (8.3-10.1 x 5.3-6.3 um, Q = 1.47 -1.78, Q =
1.60) and a solitary growth habit. Except for the mentioned species, C. amplus is also
similar to C. pakistanicus. Clavariadelphus pakistanicus, another species also from Asia,
is distinct in smaller basidiomes (up to 12 cm high), with narrower fertile apices (up to
1.4 cm), smaller basidiospores (7.5—9.2 x 4.0—5.6 um), solitary growth habit at lower
elevations and violet-brown staining reactions to KOH (Hanif et al. 2014).

In the ITS tree, C. amplus exhibits a sister relationship with C. pakistanicus with
strong support (Fig.1).

3. Clavariadelphus elongatus J. Khan, Sher & Khalid, Phytotaxa 365: 184, 2018
Figs 2d, 2e, 3c, 4c, 5c, 8a, 8b

Note. The following description is taken from Sher et al. (2018), field notes of the
Chinese material including macro-morphology, growth habit, distribution, host plants
and our examination of the specimens.

Description. Basidiomes up to 28 cm high, 0.5-1.0 cm diam. basally, enlarged
upwards to 1.5 cm diam., subcylindrical to fusiform, simple or occasionally branched,
laterally compressed in age; Aymenium longitudinally rugose, plum colour (13C2-4)
or light purple to greyish-purple (14C2-3) or dull-lilac (15D2-3); apex tapered, suba-
cute to obtuse, initially smooth, rugulose in age, caramel-brown to sandy-brown or
sienna (6C5-—6); base terete, smooth, white; mycelial hyphae scant, white; flesh initially
solid, then soft and spongy in age. Odour and taste not recorded.

Clavariadelphus species 103

Qe
10 um

Figure 8. Microscopic features of Clavariadelphus elongatus (HKAS 76589). a Leptocystidia and im-
mature basidia b Basidia.

\

Hymenium extending over the apex of the basidiomata, composed of basidia and lep-
tocystidia. Basidia 75-95 x 6-10 um, clavate, hyaline, thin-walled, 4-spored, sterigmata
7-10 um in length. Basidiospores [40/2/2] (8.3—) 9.0-11.0 (—12.0) x (5.5—) 5.7—7.4 um,
Q = (1.43-) 1.44-2.04 (-2.31), Q = 1.71 + 0.16, narrowly ellipsoid to ellipsoid, ovate or
amygdaliform, with a small apiculus, inamyloid, thin-walled, hyaline in KOH, smooth.
Leptocystidia 70-75 x 3.5-4.5 um, scattered amongst and scarcely projecting beyond the
basidia, cylindrical to narrowly clavate, thin-walled, smooth, hyaline, non-pigmented,
clamped, inflated apically at maturity, at times with apical or subapical branches. Myce-
lial hyphae 2-3 or 6-8 wm diam., interwoven or aggregated into rhizomorphic strands,
branched, clamped; the hyphal walls echinulate with light microscopy, encrusted with
massive triangular or irregular, flaky crystals up 1 um high, which are insoluble in KOH.

Chemical reactions. (dried basidiomes): KOH = positive, light yellow; FeCl, =
positive, green-yellow; NH,OH = positive, orange; ethanol, FeSO,, phenol and Melz-
ers reagent = negative.

Known distribution and ecology. NW and SW China (in this study), Pakistan
(Sher et al. 2018). Solitary to scattered on the ground in coniferous woods (Abies spp.
and Picea spp.) or mixed with broad-leaved trees (Quercus spp., Rhododendron spp. and
Salix spp.) at elevations ranging from 3000-4350 m.

Materials examined. Cuina. Gansu Province: Zhouqu Prefecture, Shatan National

Forest Park, Abies spp. woods, 16 August 2012, X.T Zhu 740 (HKAS 76589). Sichuan

104 Hong-Yan Huang et al. / MycoKeys 70: 89-121 (2020)

Province: Litang Prefecture, Gaowa, Kobresia-Bistorta meadows with extensive areas
of dwarf Rhododendron and Salix scrub with Picea spp., 30°10.10'N, 100°35.12'E, alt.
4300-4350 m, 8 August 2006, Z.W. Ge 1221 (HKAS 50801); Yajiang Prefecture, mead-
ows with shrub thickets and Picea spp. forests, 30°2.67'N, 101°18.48’E, alt. 3850-3870
m, 4 August 2006, Z. W. Ge 1162 (HKAS 50742). Yunnan Province: Yulong Prefecture,
Lizui Village, mixed coniferous and broad-leaved forests of Picea spp. and Quercus spp.,
alt. 3000 m, 23 August 2007, Y Zhang 36 (HKAS 52425); Shangri-La Prefecture,
27°29.00'N, 99°25.00'E, alt. 3600 m, 13 August 2008, 7’Z. Wei 150 (HMAS 260746).

Comments. Clavariadelphus elongatus was originally described from Pakistan (Sher
et al. 2018). In this study, it was found in NW and SW China. This species is unique in
its greyish-purple basidiomes with acute to subacute, non-enlarged apex, hyphae of the
basal mycelium encrusted with massive, flaky crystals and basidiomes having a light
yellow reaction to KOH. Clavariadelphus himalayensis, another Asian taxon, might be
confused with C. elongatus since both have a tinge of grey-purple when young. How-
ever, C. himalayensis is distinct in having smaller basidiomes, pastel-red colouration at
maturation, shorter basidiospores (8.2—-9.4 x 5.0—-5.5 um), hyphae of the basal myce-
lium covered nipple-shaped protuberances without crystals and basidiomes having a
brown-yellow reaction to KOH.

Phylogenetically, C. elongatus is related to C. pistillaris and the sequence of “C. oc-
cidentalis” from GenBank with weak support (Fig. 1).

4. Clavariadelphus gansuensis J. Zhao & L.P. Tang, sp. nov.
MycoBank No: 830272
Figs 2f, 3d, 4d, 5d, 9a, b

Diagnosis. This species is characterised by its orange, clavate basidiomes with slightly
enlarged, truncate, sterile apex, broadly ellipsoid to ellipsoid basidiospores, hyphae
of the basal mycelium with nipple-shaped protuberances and prism-like crystals and
basidiomes that turn pink or light cherry-red in KOH. It differs from C. truncatus by
the latter’s robust, darker basidiomes with enlarged apices, and larger basidiospores.
Etymology. Latin “gansuensis” refers to the holotype location in Gansu Province.
Description. Basidiomes up to 9 cm high, enlarged upwards to 1.6 cm diam., sim-
ple, clavate; hymenium longitudinally rugose, pruinose, light yellow to greyish-orange
at maturity; apex initially obtuse or broadly rounded, flattening laterally, then truncate,
slightly rugose, light orange or melon-orange (5A5—7) to orange (6AG6—7) in age; base
terete, smooth, pruinose, dirty white or pallid where covered, otherwise pruinose, pale
orange or light orange (SA3-4); mycelial hyphae white; flesh initially solid, then soft and
spongy upwards as the apex enlarges, white to pallid. Odour and taste not recorded.
Hymenium limited to the side of basidiomata, composed of basidia and leptocys-
tidia; the apex of basidiomata composed of sterile elements 15—25 x 5—7 um, clavate,
thin-walled, smooth, clamped. Basidia 75-90 x 8-10 um, clavate, hyaline, thin-walled
to thick-walled, 4-spored, sterigmata 7-10 pm in length. Basidiospores [20/1/1] 8.3-
10.1 (-10.3) x 5.3-6.3 (-6.4) um, Q = (1.34-) 1.47 -1.78 (-1.83), Q_ = 1.60 + 0.09,

Clavariadelphus species 105

—

0 um

: b (9)

10 um

Figure 9. Microscopic features of Clavariadelphus gansuensis (HKAS 76487, holotype). a Leptocystidia

and immature basidia b Basidia.

ellipsoid to broadly ellipsoid, ovate or amygdaliform, with a small apiculus, inamyloid,
thin-walled, hyaline in KOH. Leptocystidia 50-65 x 3-5 um, scattered amongst and
scarcely projecting beyond the basidia, cylindrical to narrowly clavate, thin-walled,
smooth, hyaline, non-pigmented, clamped, inflated apically at maturity, at times with
apical or sub-apical branches. Mycelial hyphae 2-3 ym diam., interwoven or aggregated
into rhizomorphic strands, branched, clamped; the hyphal walls echinulate with light
microscopy, covered with massive nipple-shaped protuberances, as well as encrusted
with prism-like crystals up 5 um long that are insoluble in KOH.

Chemical reactions. (dried basidiomes): KOH = positive, pink, light coral or light
cherry-red; FeCl, = positive, green-yellow; NH ,OH = positive, golden-rod or vivid yel-
low; phenol = positive, yellow; ethanol, FeSO ; and Melzer’s reagent = negative.

Known distribution and ecology. NW China, Gansu Province. Solitary on the
ground in coniferous woods (Abies spp.) or mixed with broad-leaved trees (Betula spp.
and Rosaceae) at elevations of approximately 3000 m.

Materials examined. Cuina. Gansu Province: Lintan Prefecture, Yeliguan Nation-
al Forest Park, coniferous woods (Abies spp.) or mixed with Betula spp. and Rosaceae
plants, alt. 3000 m, 10 August 2012, X. 77 Zhu 638 (HKAS 76487, Holotype); Wudu
Prefecture, September 1992, ML. Tian M6465 (HMAS 63052).

Comments. Clavariadelphus gansuensis, currently known only from NW China,
is distinct by its slender, clavate, orange basidiomes with truncate apex, ellipsoid ba-

sidiospores (8.3—10.1 x 5.3-6.3 um), pink staining reaction to KOH, hyphae of the

106 Hong-Yan Huang et al. / MycoKeys 70: 89-121 (2020)

basal mycelium with nipple-shaped protuberances and prism-like crystals and solitary
growth habit in coniferous or mixed forests.
This species is most likely to be confused with several taxa, including C. amplus,
C. pallido-incarnatus, C. pakistanicus, C. truncatus and C. unicolor. The comparison
between C. gansuensis and C. amplus can be found in our treatment of C. amplus.
According to our phylogenetic analyses, C. gansuensis is allied with the sequence of
“C. truncatus’ from GenBank with strong support (Fig. 1).

5. Clavariadelphus himalayensis Methven, Mem. New York Bot. Garden 49: 152, 1989
Figs 2g, 3e, 4e, f, 5e, 10a, b

Note. The following description is mainly from Methven (1989), combined with our
field notes, including macro-morphology, growth habit, distribution, host plants and
examination.

Description. Basidiomes up to 15 cm high, 1-1.5 cm diam. basally, slightly en-
larged towards to 2 cm diam., simple, narrow clavate, ligulate to spathulate, laterally
compressed in mature specimens; /ymenium initially smooth, longitudinally rugose in
age, greyish-red to pastel-red; apex obtuse, smooth, concolorous with the hymenium;
surface not staining where cut or bruised; base terete, smooth, pruinose, pallid-white;
mycelial hyphae interwoven, white to pallid; flesh soft and spongy, hollow apically in
age, white to cream colour, not staining on exposure. Odour and taste not recorded.

oO

10 um

Figure 10. Microscopic features of Clavariadelphus himalayensis (HKAS 58811). a Leptocystidia and

immature basidia b Basidia.

Clavariadelphus species 107

Hymenium extending over the apex of basidiomata, composed of basidia and lepto-
cystidia. Basidia 75-95 x 8-11 um, clavate, hyaline, thin-walled, (2—) 4-spored, sterig-
mata 8—10 ym in length. Basidiospores [20/1/1] (7.8—) 8.2—9.4 (—9.6) x (4.6—) 5.0-5.5
(6.0) um, Q = 1.50-1.82 (-1.90), Q_ = 1.56 + 0.08, ellipsoid to broadly ellipsoid or
ovate, with a small apiculus, inamyloid, thin-walled, hyaline in KOH, smooth. Lep-
tocystidia 50-70 x 2.5-3.5 um, scattered amongst and scarcely projecting beyond the
basidia, cylindrical to narrowly clavate, thin-walled, smooth, hyaline, non-pigmented,
clamped, inflated apically at maturity, at times with apical or subapical branches. Myce-
lial hyphae \—2 or 3—5 pm diam., interwoven or aggregated into rhizomorphic strands,
branched, clamped; walls thin or irregularly slightly thickened, the hyphal walls echi-
nulate under light microscopy, covered nipple-shaped protuberances with SEM.

Chemical reactions. (dried basidiomes): KOH = positive, golden-yellow; FeCl, =
positive, green-yellow; NH {OH = positive, orange; ethanol, FeSO te Melzer’s reagent
and phenol = negative.

Known distribution and ecology. SW China (in this study) and India (Methven
1989). Solitary to gregarious habit on the ground in mixed woods at elevations above
3200 m.

Materials examined. Cuina. Yunnan Province: Shangri-La Prefecture, mixed co-
niferous (Pinus spp.) and broad-leaved forests (Caragana spp., dwarf Quercus moni-
motricha and Sanguisorba spp.), 27°28.55'N, 99°53.05'E, alt. 3280 m, 27 June 2006,
Z.W. Ge 1113 (HKAS 50684). Lijiang Prefecture, mixed conifers, alt. 3300 m, 27
August 2009, Q. Cai 146 (HKAS 58811).

Comments. Clavariadelphus himalayensis was originally described from India
(Methven 1989). It is the first report from China. Chinese collections match the origi-
nal descriptions except for slightly smaller basidiospores (8.2—9.4 x 5.0—5.5 um). The
difference in basidiospore size might be from measurement error or the collections
being from different geographical regions. Clavariadelphus himalayensis is distinct by
its pastel-red to greyish-red, ligulate to spathulate basidiomes flesh that does not stain
where bruised or cut, broadly ellipsoid basidiospores (9-11 x 5—6 um from the holo-
type; Methven 1989), hyphae of the basal mycelium with nipple-shaped protuberances
and a negative reaction with phenol. Other taxa from Asia, which might be confused
with C. himalayensis include C. mirus (Pat.) Corner and C. yunnanensis. Although
similar in size to those of C. himalayensis, the basidiomes of C. mirus are light brown
to brown and produce broadly ovate, larger basidiospores (10-13 x 6-8 um; Methven
1990). Clavariadelphus yunnanensis, known from northern India and SW China, is
distinct by its larger basidiomes that are light brown, larger basidiospores (10—13.5 x
6.5—8 um), hyphae of the basal mycelium covered by massive nipple-shaped protuber-
ances and a light yellow staining reaction with phenol. Additionally, the flesh of C.
himalayensis does not stain where bruised or cut, whereas the flesh of C. mirus and C.
yunnanensis slowly stains brunnescent to russet on exposure.

The phylogenetic analyses show that C. himalayensis is allied with the sequence of
“C. pistillaris’ and Clavariadelphus (JQ991679 from Zhejiang Province, China) from
GenBank with weak support (Fig. 1). More data are needed for understanding the
phylogenetic relationship of the three species.

108 Hong-Yan Huang et al. / MycoKeys 70: 89-121 (2020)

6. Clavariadelphus khinganensis J. Zhao, L.P. Tang & P. Zhang, sp. nov.
MycoBank No: 830273
Figs 2h-i, 3f, 4g, lla, b

Diagnosis. This species is distinct from other taxa in Clavariadelphus by the yellowish-
brown, clavate basidiomes with slightly enlarged apex, narrowly ellipsoid basidiospores
and basidiomes that turn very light yellow in KOH.

Etymology. Latin “khinganensis” refers to the holotype location, Greater Khingan
Mountains or Da Xing’an Ling, in NE China.

Description. Basidiomes up to 12.5 cm high, around 0.8 cm diam. basally, 2.5 cm
diam. apically, simple, initially subcylindrical to subfusiform, enlarged upwards in age,
then clavate to broadly clavate, finally irregularly laterally compressed; hymenium ini-
tially smooth, longitudinally rugose to rugulose in age, pale yellow-brown (4A3) or pale
orange (5A 4-6) to greyish-orange (5B4—5, 6B4—5); apex obtuse or broadly rounded,
rugose, concolorous with the hymenium at maturity; base terete, smooth, white to pallid
when covered, otherwise pale yellow (4A4—5) to light orange (5A4—6); mycelial hyphae
interwoven, white; flesh initially solid, becoming soft and spongy upwards as the apex
enlarges in age, dirty white. Odour and taste not recorded. Spore deposit not recorded.

Hymenium extending over the apex of basidiomata, composed of basidia and lep-
tocystidia. Basidia 85-105 x 8-11 um, clavate, hyaline, thin-walled, 4-spored, sterig-
mata 9-10 um in length. Basidiospores [20/1/1] 9.2-12.0 x 4.6-6 um, Q = 1.6-2.2,
Q = 1.97 + 0.17, narrowly ellipsoid or amygdaliform, with a small apiculus, inamy-
loid, thin-walled, hyaline in KOH, smooth. Leptocystidia 60-70 x 3-4 um, scattered
amongst and scarcely projecting beyond the basidia, cylindrical to narrowly clavate,
thin-walled, smooth, hyaline, non-pigmented, clamped, inflated apically at maturity,
at times with apical or subapical branches. Mycelial hyphae \acking material.

Chemical reactions. (dried basidiomes): KOH = positive, very light yellow; etha-
nol, FeCl, FeSO, phenol, Melzer’s reagent and NH {OH = negative.

Known distribution and ecology. N China. Solitary on the ground in broad-
leaved forests at around 800 m altitude.

Materials examined. CHINA. Jilin Province: Antu Prefecture, Er-dao-bai-he
Town, Changbai Mountains, mainly broad-leaved forests (Betula platyphylla, Cory-
lus mandshurica, and Quercus monimotricha), mixed with the coniferous tree (Pinus
koraiensis), 42°24.05'N, 128°6.00'E, alt. 753 m, 18 August 2019, H.Y Huang 368
(MHKMU H.Y. Huang 368). Inner Mongolia: De-er-bu-er Town, Greater Khingan
Mountains, alt. 800 m, 6 August 2013, P Zhang 1289 (MHHNU 7789 Holotype);
Ku-ti-he Town, Zha-lan-tun City, 24 July 1985, W Huang s. n. (HMAS 49920).

Comments. Clavariadelphus khinganensis, known from broad-leaved forests in N
China, is distinct by its solitary habit at low elevations (around 800 m), small size, pale
brown-orange basidiomes, ellipsoid basidiospores and very pale yellow reaction in KOH.

Morphologically, C. khinganensis is quite similar to two Asian taxa, C. mirus and C.
yunnanensis. However, C. mirus was originally described from northern Vietnam and
has larger basidiomes, broader basidiospores and a tropical distribution (Butan, India,

Clavariadelphus species 109

b 10 ym

Figure 11. Microscopic features of Clavariadelphus khinganensis (MHHNU 7789, holotype). a Lepto-

cystidia and immature basidia b Basidia.

Nepal; Methven 1990). Clavariadelphus yunnanensis is unique in its habit, growing
with conifers at high elevations (above 3000 m), has darker colouration and larger
basidiomes (up to 20 cm high), broader basidiospores and basidiomes with yellow
reactivity in KOH.

Interestingly, C. khinganensis is clustered with a collection labeled as “C. truncatus”
from Canada, the GenBank accession DQ097871 (Durall et al. 2006) and there are
no genetic differences on ITS (Fig. 1). It indicates C. khinganensis may be distributed
in Canada. More data from North America are needed to confirm the distribution
pattern of this species. The sister relationship of C. khinganensis cannot be resolved
according to the present data.

7. Clavariadelphus ligula (Schaeff.) Donk, Rev. Niederl. Homob. Aphyll. 2:73, 1933
Figs 3g, 12a, b

Note. The following taxonomic description is drawn from Methven (1990) and our
observations.

Description. Basidiomes up to 10 cm high, 0.2—0.8 cm diam. basally, slightly en-
larged upwards, simple, narrowly clavate to clavate; hymenium longitudinally rugose in
age, light yellow, brownish-orange to light brown at maturity; apex subacute to obtuse
or broadly rounded, surface slightly rugulose, concolorous with the hymenium; surface

110 Hong-Yan Huang et al. / MycoKeys 70: 89-121 (2020)

10 um

Figure 12. Microscopic features of Clavariadelphus ligula (HMAS 35954). a Leptocystidia and imma-
ture basidia b Basidia.

slowly staining brownish-orange to brownish-grey where cut or bruised; base terete,
initially pale yellow to light yellow, then brownish-orange to light brown to brown;
mycelial hyphae white to pallid; flesh initially solid, becoming soft and spongy upwards
as the apex enlarges in age, white to pallid. Odour not distinctive. Taste not distinctive
or slightly sweet. Spore deposit yellowish-white to light buff in mass.

Hymenium extending over the apex of basidiomata, composed of basidia and lep-
tocystidia. Basidia 45-85 x 8-11 um, clavate, hyaline, thin-walled, 4-spored, sterigma-
ta 9-10 pm in length. Basidiospores 11.0-14.0 x 4.0-5.5 um, Q = 2.4-3.1, Q_ = 2.7,
narrowly ellipsoid, with a small apiculus, inamyloid, thin-walled, hyaline in KOH,
smooth. Leptocystidia 40-80 x 2.5—5 um, scattered amongst and scarcely projecting
beyond the basidia, cylindrical to narrowly clavate, thin-walled, smooth, hyaline, non-
pigmented, clamped, inflated apically at maturity, at times with apical or subapical
branches. Mycelial hyphae 2-4 um diam., interwoven or aggregated into rhizomorphic
strands, branched, clamped. Insufficient material to perform SEM.

Chemical reactions. (dried basidiomes): KOH = positive, lemon-chiffon; NH,OH =
positive, orange; ethanol, FeCl,, FeSO,, Melzer’s reagent and phenol = negative.

Known distribution and ecology. Widespread in the Northern Hemisphere, in-
cluding in North America, Bulgaria, NE China, England, Estonia, Finland, Germany,
India, Italy, Sweden and Switzerland (Methven 1990). Scattered to gregarious habit on
the ground in mixed woods (Abies, Picea, Pinus, Thuja and Tsuga).

Clavariadelphus species i

Materials examined. Cuina. Heilongjiang Province: Linkou Prefecture, 19 Au-
gust 1972, X.L. Mao, s. n. (HKAS 35954); same location, Q.X. Wu, s. n. (HMAS
51688). Czecu: 2 September 1960, MZ. Geesteranus 13290 (HMAS 41146).

Comments. Clavariadelphus ligula was originally described from Germany, but
was also reported in China (Mao 2009). Our study confirms that this species is mainly
found in N China, whereas our data do not support the previous report of the dis-
tribution in SW China (Mao et al. 1993; Mao 2009). The basidiospores of Chinese
collections (11.0—14.0 x 4.0-5.5 um, Q = 2.4—3.1, Q. = 2:7 vate smaller and broader
than the neotype of C. ligula from Germany (12.0-16.5 x 3.5—-4.5 um, Q = 2.9-4.6,
Q_ = 3.7; Methven 1990).

Morphologically, C. ligula and C. sachalinensis are similar in the field. However,
C. sachalinensis has more elongated, narrower basidiospores (21-24 x 4-6 um, Q =
3.5-5.0, Q. = 4.2). Additionally, C. ligula lacks any reaction with FeCl,, whereas C.
sachalinensis turns green-yellow in FeCl,. Clavariadelphus yunnanensis is likely to be
confused with C. /igula when young. However, C. yunnanensis differs in that it has
larger basidiomes (up to 20 cm high), smaller and broader basidiospores (9.0-11.0 x
4.6-6.4 um, Q = 1.32-1.72, Q_ = 1.56) and a positive reaction with phenol.

The phylogenetic analyses show that C. /igula is allied with the sequence of C.
americanus from GenBank with strong support (Fig. 1).

8. Clavariadelphus sachalinensis (S. Imai) Corner, Ann. Bot. Mem. I: 282, 1950
Figs 2); 3H, 5f;'¢°13a,'b

Note. The following taxonomic description is drawn from Methven (1989), combined
with our field notes, including macro-morphology, growth habit, distribution, host
plants and observations.

Description. Basidiomes up to 8 cm high, 0.3-0.6 cm diam. basally, slightly en-
larged upwards 0.8—1.2 cm diam., simple, initially cylindrical to subcylindrical, then
narrowly clavate to clavate; hymenium longitudinally rugose in age, tawny or light
walnut-brown to light brown at maturity; apex subacute, obtuse to broadly rounded,
surface smooth to slightly rugulose, concolorous with the hymenium; surface slowly
staining, brown or dark brown where cut or bruised, staining more conspicuously; base
terete, pubescent to tomentose, initially pale yellow to light yellow, then brownish-
orange to light brown; mycelial hyphae greyish to pallid; flesh initially solid, becoming
soft and spongy upwards, white to pallid, staining on exposure. Odour and taste not
distinctive. Spore deposit yellowish-white to light buff.

Hymenium extending over the apex of basidiomata, composed of basidia and leptocys-
tidia. Basidia 65-105 x 8-12.5 um, clavate, hyaline, thin-walled, (2—) 4-spored, sterigma-
ta 8-10 um in length. Basidiospores 21-24 x 4-6 um, Q = 3.55.0, Qe= 4,2, narrowly el-
lipsoid, boletoid or sway-backed in profile, with a small apiculus, inamyloid, thin-walled,
hyaline in KOH, smooth. Leptocystidia 50-70 x 2.5—5 um, scattered amongst and scarcely
projecting beyond the basidia, cylindrical to narrowly clavate, thin-walled, smooth, hya-

112 Hong-Yan Huang et al. / MycoKeys 70: 89-121 (2020)

10 um 10 um

Figure 13. Microscopic features of Clavariadelphus sachalinensis (MHHNU 7816). a Basidia b Lepto-

cystidia and immature basidia.

line, non-pigmented, clamped, inflated apically at maturity, at times with apical or subapi-
cal branches. Mycelial hyphae 2-8 um diam., interwoven or aggregated into rhizomorphic
strands, branched, clamped; hyphal walls smooth with light microscopy and SEM.

Chemical reactions. (dried basidiomes): KOH = positive, light yellow; FeCl, =
positive, green-yellow; NH,OH = positive, orange; ethanol, phenol, FeSO, and Melz-
ers reagent = negative.

Known distribution and ecology. N China (in this study) and SW China (Meth-
ven 1990). Gregarious habit on fallen needles and other debris under conifers, espe-
cially pine at elevations ranging from 2000-3600 m.

Materials examined. Cuina. Inner Mongolia: Mo er dao ga National Forests,
Great Khingan Mountains, 8 August 2013, P Zhang 1316 (MHHNU 7816). Sichuan
Province: Hongyuan Prefecture, Kangle Town, alt. 3600 m, 14 August 1998, M.S.
Yuan 3361 (HKAS 33844).

Comments. Clavariadelphus sachalinensis was proposed by Imai, based on Japa-
nese collections as a species of Clavaria and then transferred to genus Clavariadelphus
(Imai 1930, Corner 1950). In China, C. sachalinensis was previously reported with
distribution in SW China (Methven 1990; Zang 1996) and is also found in northern
regions of China. Clavariadelphus sachalinensis is similar to C. ligula and C. yunnanen-
sis. Their differences are described in our discussion of C. ligula.

Clavariadelphus species |

9. Clavariadelphus yunnanensis Methven, Mem. New York Bot. Garden 49: 156 1989
Figs 2j-l, 3i, 4h, 5h, 14a, b

Note. The following taxonomic description is mainly drawn from Methven (1989).
Field notes including macro-morphology, growth habit, distribution and host plants,
SEM characteristics and chemical tests are from this study.

Description. Basidiomes up to 20 cm high, 0.5 cm diam. basally, enlarged upwards
2 cm diam., simple, initially cylindrical to subcylindrical, then narrowly clavate, sub-
olanceolate; ymenium initially smooth, longitudinally rugose to rugulose in age, light
brown to cinnamon at maturity; apex obtuse, smooth to rugose, concolorous with the
hymenium; surface slowly staining, russet to umber; dase terete, smooth, pale cinna-
mon or pale ochraceous-buff; mycelial hyphae white; flesh initially solid, becoming soft
and spongy upwards as the apex enlarges, white to pinkish-buff. Odour not distinctive.
Taste slightly bitter. Spore deposit white.

Hymenium extending over the apex of the basidiomata, composed of basidia and
leptocystidia. Basidia 70-80 x 8-9 um, clavate, hyaline, thin-walled, (2—) 4-spored,
sterigmata 7-10 um in length. Basidiospores [40/2/2] (8.8—-) 9.0-11.0 x 4.6-6.4
(-7.4) um, Q = (1.29) 1.32-1.72 (-1.76), Q_ = 1.56 + 0.11, ellipsoid to broadly el-
lipsoid, ovate or amygdaliform, smooth. Leptocystidia 40-60 x 2.5—3.5 um, scattered
amongst and scarcely projecting beyond the basidia, cylindrical to narrowly clavate,
thin-walled, smooth, hyaline, non-pigmented, clamped, inflated apically at maturity,

at times with apical or subapical branches. Mycelial hyphae 2-4 um diam.., parallel,

Figure 14. Microscopic features of Clavariadelphus yunnanensis (HKAS 57731). a Leptocystidia and

immature basidia b Basidia.

114 Hong-Yan Huang et al. / MycoKeys 70: 89-121 (2020)

interwoven or aggregated into rhizomorphic strands, branched, clamped; walls thin or
irregularly slightly thickened, the hyphal walls echinulate with light microscopy, cov-
ered with massive nipple-shaped protuberances and lacking crystals with SEM.

Chemical reactions. (dried basidiomes): KOH = positive, golden-yellow; FeCl, =
positive, green-yellow; NH,OH = positive, golden-rod or vivid yellow; phenol = posi-
tive, light yellow; ethanol, FeSO, and Melzer’s reagent = negative.

Known distribution and ecology. SW China and northern India (Methven
1989). Either solitary, scattered or gregarious habit on the ground in mixed deciduous-
coniferous forests in association with several genera (e.g. Abies, Berberis, Picea, Pinus,
Quercus, Rosa and Salix) at elevations ranging from 2200-3600 m.

Materials examined. Cutna. Sichuan Province: Hongyuan Prefecture, Shua-
jing Temple, Picea, alt. 3400 m, 3 August 1996, M.S. Yuan 2375 (HKAS 30752);
Kangding Prefecture, Liuba, alt. 3500 m, 9 September 1996, M.S. Yuan 2686 (HKAS
31136); Kangding Prefecture, Zheduo Mountains, shrubs dominated by Berberis,
Quercus, Rosa, Salix, alt. 3585 m, 14 August 2008, Z.W Ge 903 (HKAS 49398).
Yunnan Province: Shangri-La Prefecture, 19 August 2008, 28°18.00'N, 98°33.00'E,
alt. 3100 m, 7'Z. Wei 270 (HMAS 250510); Deging Prefecture, Xiaruo, 18 Septem-
ber 2010, HBB2010-D15 (HKAS 62644); Jianchuan Prefecture, Shibao Mountains,
14 August 2003, Z.W. Ge 4 (HKAS 43816); same location, 30 August 2009, G. Wu
199 (HKAS 57731); Kunming City, Yeya Lake, alt. 2200 m, 22 September 2012,
Z.L. Yang 5629 (HKAS 77288); Shangri-La Prefecture, Haba Mountains, 13 August
2008, L.P Tang 618 (HKAS 54849); Shangri-La Prefecture, 16 August 2008, 7'Z.
Wei 271 (HMAS 250466); Shangri-La Prefecture, Bita Lake, 24 August 2009, Q. Cai
122 (HKAS 58789); same location and date, G. Wu 127 (HKAS 57659); Weixi Pre-
fecture, Qizong, 19 September 2010, HBB2010-W21 (HKAS 61417); Yulong Prefec-
ture, Yulong Snow Mountains, Sandaowan, under Adies spp., alt. 3200 m, 1 August
1995, M. Zang 12514 (HKAS 30038); Yulong Prefecture, Lizui Village, 20 August
2008, Q. Zhao 8262 (HKAS 55244); Yulong Prefecture, Jiuhe, 20 August 2010, G.
Wu 327 (HKAS 63558); Yulong Prefecture, Yulong Snow Mountains, Ganhaizi, under
Picea spp., alt. 3100 m, 3 September 1986, VM. Zang 10739 (HKAS 17788); Yulong
Prefecture, Yulong Snow Mountains, Yu Lake, Adzes forests, alt. 3000 m, 1 August
1985, M. Zang 10220 (HKAS 15063); Yulong Prefecture, Yulong Snow Mountains,
6 September 1986, R.H. Petersen s. n. (HKAS 20067); same location and date, RZ.
Petersen s. n. (HKAS 20068); Yulong Prefecture, Wenhai, 17 September 2012, G. Wu
1054 (HKAS 77226).

Comments. Clavariadelphus yunnanensis is quite common in SW China where it
was previously reported as C. ligula or C. pistillaris (Mao et al. 1993; Yuan and Sun
1995; Zang 1996; Mao 2009). It is well characterised by its cinnamon buff, large ba-
sidiomes, broadly ellipsoid basidiospores, hyphae of the basal mycelium covered with
nipple-shaped protuberances and occurrence at high elevation forests. This taxon is
also similar to C. ligula and C. sachalinensis, but differs microscopically in the size
and shape of the basidiospores (see the comments under C. /igu/a). Immature fruit
bodies of C. yunnanensis are similar to C. griseoclavus. However, the latter can be dis-

Clavariadelphus species 115

tinguished from smaller basidiomata (less than 13 cm high), narrower apex (less than
1.5 cm diam.) and narrower basidiospores (Q_ 1.89) (Lu and Li 2020). Although C.
yunnanensis might be confused with the Asian taxon C. mirus, the latter is distinct by
its slender cylindrical, light brown basidiomes and broader basidiospores (Methven
1990). The presence of C. mirus in China needs to be ascertained. In the phylogenetic
analyses, C. yunnanensis has a joint relationship with C. elongatus, C. pistillaris and
the sequence of “C. occidentalis” from Tunisia, but the sister relationship cannot be

resolved (Fig. 1).

Taxonomic key to species of Clavariadelphus in China

i Basidiesporesinartowly cliipsoid, Ois2 11a). tee. samtens oransesugee: stuns 2
- Basidiospores broadly ellipsoid to ellipsoid, Q < Qeeecceceseeeeeeeeeeeeeeeens 3
2, Basidiospores 11.0—14.0 x 4.0-5.5 pm, Q_ 2.7 ..sccsssreresrsrercneseerones C. ligula
- Basidiospores 21-24 x 4-6 pin; 27 A.D coscveecsesseoncesnernceteine C. sachalinensis
3 Basidignaes Ora ce; apex sterile) 1 PUN CAbe sa... secakaounoa caencanatcdan csenteinaristene scteaeh A
~ Basidiomes without orange tinge; apex fertile, not truncate wo... eee 5
4 Basidiomata-apex: 3=7-5 Cit Gia finden nsentenote ett buen epee C. amplus
- Basidiomata apex. 2 Cnr dianicy 0.) cn csoteenorcternenee spe sauoneetas C. gansuensis
5 Basichorives tug tally;- 209 Osriy O Mctaaddeitnaicsstnniaiehhalecsaniteassbieiedehed einen laabas 6
- Basidionesaisually: 2-2 0cCHir MG se acerstese APteos sxaearrtresein sapsn tease counatentns Pewee? "i
6 Basidiomes grey-purple; basidiospores narrowly ellipsoid, 9.0-11.0 x 5.7—
TAMIR ML UTEN Oasu As Neratiohcastisntortongsbatan Siaminoibecsstterncskatene nate C. elongatus
- Basidiomes cinnamon; basidiospores broadly ellipsoid, 9.0-11.0 x 4.6-6.4
Wf SIN TDG a sR capescen tsutyoa nants onned gs asiiva an eias Uieiginde Gn Tea eer C. yunnanensis
7 Basidiomes greyish-red to pastel-red...... ieee eeeeeeseeeeeeeeees C. himalayensis
— Basidiomes grey or yellow, without red colouration... eee eeeeeeeeeeeeeee 8
8 Basidiomes grey; basidiospores ellipsoid 10-11 x 5—6.5 um, (OAs petra
Litialliales chads dahegarastinentart oonhl nati hated aenana tbe samtaeaehPalbeatensa Sedat ees C. griseoclavus
Basidionaes vellownce lotratl O11, is wats dasetvndcaes onaesieyndedipeedde sebeaesintgereneboodeonkeee 9
9 Basidiomes yellow; basidiospores broadly ellipsoid 7.8-9.6 x 5.5—7.4 um,
COPE, DSi saa tea teas asec cbbage needa aso sebedbieasst rin pele teladnpsntes Ment Fessebneny C. alpinus
a Basidiomes pale yellow-brown; basidiospores narrowly ellipsoid 9.2—12.0 x
AOL MEINE Tre oc eereate icon easier iene Cexe'ivecenn ovine teen C. khinganensis
Discussion

The taxonomic importance of comprehensive data in Clavariadelphus

Many studies have verified that molecular methods are effective in resolving relation-
ships in complicated groups of fungi (Zeng et al. 2013; Tang et al. 2017; Huang et al.

116 Hong-Yan Huang et al. / MycoKeys 70: 89-121 (2020)

2018; Yang et al. 2018). In a pre-study analysis, we evaluated four DNA gene markers:
ITS, large subunit of nuclear ribosomal RNA (nrLSU), translation elongation factor
lo gene (tefl-a) and DNA-directed RNA polymerase II second subunit (7pb2). Com-
pared to the others, ITS offered the highest probability of successful identification
in Clavariadelphus. By contrast, other markers displayed a lower success rate of PCR
amplification or inferior species resolution in some close or sibling taxa. ITS sequences
acquired from this study are listed in Appendix 1.

Macro-morphological, micro-morphological and SEM characteristics are very
important in the taxonomy of Clavariadelphus. Although Clavariadelphus can be
readily distinguished from other clavarioid genera, the delimitation of infrageneric
taxa is difficult in many cases, especially without critical observation and examination
(Methven 1990). Basidiomata colour is a diagnostic characteristic, although it must
be used in conjunction with other morphological features. In China, basidiomata
colour ranges from yellow to orange, grey-purple, pastel-red or brown. Despite the
inherent variability in shape, size and other characteristics of the basidiomes, these
features are important diagnostic characteristics in Clavariadelphus, as well as the size
and shape of basidiospores. The basidiospores of Chinese taxa of this genus are sum-
marised in Fig. 3 and 4. Additionally, some SEM characteristics, especially hyphae of
the basal mycelium, are of taxonomic value. The variations of basal hyphae of Chinese
taxa range from smooth, having nipple-shaped protuberances to crystals or both at
the same time (Fig. 5).

Chemical reactions also are helpful in distinguishing Clavariadelphus species. Doty
(1948) distinguished this genus using FeSO, reactions. Corner reported that chemi-
cal reactions, especially KOH, are useful for delimiting Clavariadelphus taxa (Corner
1950). In this study, chemical colour reactions were conducted using seven chemical
reagents (Table 1). Amongst these, four reagents were found to be discriminatory with
some species, specifically KOH, FeCl, NH,OH and phenol. Three additional reagents,
namely ethanol, Melzer’s reagent and FeSO yp were found to lack discriminatory value.

Metadata supply taxonomic information, such as habit, distribution and host
plants. The growth habit of Chinese taxa includes solitary, scattered and gregarious.
Growth habit is of taxonomic value only when used in conjunction with other features
(Methven 1990). The Chinese specimens were collected in mixed or coniferous forests
in association with Abies, Berberis, Quercus, Pinus, Picea, Rhododendron, Rosa, Salix,
Thuja and Tsuga. The distribution of a species usually correlates with that of its host
plant. Although the Chinese taxa exhibit no apparent preference of host plants, the
so-called cosmopolitan species within Clavariadelphus seem to be rare in this study.

Clavariadelphus species diversity in China

Many new fungal taxa have been discovered in the last ten years in China (Zhang et
al. 2005; Zeng et al. 2013; Tang et al. 2014; Huang et al. 2018; Yang et al. 2018).
However, there are still a large number of undescribed fungal taxa in this country.
This study indicates that there are at least ten known taxa of Clavariadelphus in China,

Clavariadelphus species LF

including four previously described (C. griseoclavus, C. ligula, C. sachalinensis and C.
yunnanensis), two not previously reported in China (C. elongatus and C. himatayensis)
and four novel species (C. alpinus sp. nov., C. amplus sp. nov., C. gansuensis sp. nov.
and C. khinganensis sp. nov.). Several taxa, previously reported from China, need to
be confirmed, including C. mirus, C. pistillaris and C. truncatus. In China, there are
still some species that need to be discovered, such as GenBank accession JQ991679.
To date, with the four novel taxa described in this study, there are twenty-eight species
of Clavariadelphus worldwide. Although the taxonomy of Clavariadelphus has received
much attention in the past, this group needs to be further examined with molecular
methods. More reliable sequence data, especially those species from North America
and Europe, are needed to understand phylogenetic relationships better.

Acknowledgements

We thank the curators and collectors of the herbarium of HMAS and HKAS. Fi-
nancial support for fieldwork and experiment expenses were from the following pro-
jects: the National Natural Science Foundation of China (Nos. 31960007, 31560004,
31670015, 31670024), The Program Innovative Research Team in Science and
Technology in Yunnan Province (No. 2019HC008), Yunnan Applied Basic Research
Projects-joint special project (No. 2014FB016), the Science Research Foundation of
Department of Education, Yunnan Province (No. 2015Y147), the Program Innovative
Research Team in Science and Technology in Yunnan Province (No. 2019HC008) and
the Open Research Foundation of Yunnan Key Laboratory of Pharmacology for Natu-
ral Products (No. 2015G003). We are greatly indebted to Dr. Andrew S. Methven and
other anonymous reviewers for their valuable suggestions and comments. Many thanks
are also due to the linguistic assistance provided by TopEdit (www.topeditsci.com)
during the preparation of this manuscript.

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Appendix |

Sequences used or produced in our phylogenetic analyses of Clavariadelphus in China.

Taxon Voucher Locality GenBank Accession
No. (ITS)

Clavariadelphus alpinus HKAS 57396 China, Yunnan MK705888*
C. americanus MycoMap # 1288 USA, Indiana MK575228

C. amplus HKAS 76577 China, Gansu MK705851*
C. amplus HKAS 54876 China, Yunnan MK705857*
C. amplus HMAS 132008 China, Qinghai MK705852*
C. amplus HMAS 97090 China, Qinghai MK705853*
C. amplus HKAS 49229 China, Sichuan MK705854*
C. amplus HKAS 49278 China, Sichuan MK705855*
C. amplus HKAS 53797 China, Sichuan MK705856*
C. amplus HMAS 250466 China, Yunnan MK705858*
C. amplus HMAS 97248 China, Tibet MK705859*
C. amplus HMAS 59867 China, Tibet MK705860*
C. amplus HKAS 46160 China, Tibet MK705861*
C. amplus HKAS 46120 China, Tibet MK705862*
C. elongatus HKAS 76589 China, Gansu MK705842*
C. elongatus HKAS 50742 China, Sichuan MK705843*
C. elongatus HKAS 50801 China, Sichuan MK705844*
C. elongatus HMAS 260746 China, Yunnan MK705845*
C. elongatus HKAS 52425 China, Yunnan MK705846*
C. elongatus LAH 31397 Pakistan, Khyber Pakhtunkhwa MG768847*
C. elongatus SWAT 000559 Pakistan, Khyber Pakhtunkhwa MG768848*
C. gansuensis HKAS 76487 China, Gansu MK705847*
C. griseoclavus BJTC FM964 China, Shanxi MT302370

C. griseoclavus BJTC FM965 China, Shanxi MT302371

Taxon

C. himalayensis
C. himalayensis
C. khinganensis
C. khinganensis
C. ligula

C. ligula

C. ligula

C. mucronatus
C. occidentalis
C. occidentalis
C. occidentalis
C. occidentalis
C. occidentalis
C. pistillaris

C. pistillaris

C. pakistanicus
C. pakistanicus
C. sachalinensis
C. sachalinensis
C. sachalinensis
C. sachalinensis
C. sachalinensis

Clavariadelphus sp.
Clavariadelphus sp.
Clavariadelphus sp.
Clavariadelphus sp.
Clavariadelphus sp.
Clavariadelphus sp.

C. subfastigiatus
C. subfastigiatus
C. truncatus

C. truncatus

C. truncatus

C. truncatus

C. unicolor

C. yunnanensis
C. yunnanensis
C. yunnanensis
C. yunnanensis
C. yunnanensis
C. yunnanensis
C. yunnanensis
C. yunnanensis
C. yunnanensis
C. yunnanensis
C. yunnanensis
C. yunnanensis
C. yunnanensis
C. yunnanensis
C. yunnanensis
C. yunnanensis
C. yunnanensis
C. yunnanensis
C. yunnanensis
C. yunnanensis
C. yunnanensis
Lentaria byssiseda
Kavinia himantia
K. alboviridis

* indicates sequences generated in this study, fontbold indicates type material for the new species.

Clavariadelphus species

Voucher

HKAS 50684
HKAS 58811
MHHNU 7789
MHKMU EY. Huang 368
HMAS 51688
HMAS 35954
HMAS 41146
OSC 1064138
OSC 104664
OSC 112861
OSC 114250
OSC 114281
H21536
NAMA 2017-123
AMB 18611
MH 129901
SR1742
MHHNU 7816
p06li
p059i
p058i
OSC 96213
src121
OSC 105674
HC-PNNT-268
ECM54
Mushroom Observer.org/254047
Montri-108
OSC 119587
MICH 73554
MA-Fungi 48062
OUC9I9I108
SIM278
AMB 18612
Mushroom Observer #112193
HKAS 49398
HKAS 31136
HKAS 54849
HKAS 63558
HKAS 57731
HKAS 58789
HKAS 55244
HMAS 250510
HMAS 250471
HKAS 62644
HKAS 61417
HKAS 43816
HKAS 30752
HKAS 30083
HKAS 20068
HKAS 20067
HKAS 17788
HKAS 15063
HKAS 57659
HKAS 77226
HKAS 77288
TENN 61159
CFMR:DLL2011-079
CFMR:DLL2011-131

Locality

China, Yunnan
China, Yunnan
China, Inner Mongolia
China, Jilin
China, Heilongjiang
China, Heilongjiang
Czech, —

USA, Oregon
USA, the Pacific Northwest
USA, the Pacific Northwest
USA, the Pacific Northwest
USA, the Pacific Northwest
Tunisia, Ain Draham
USA, Wisconsin
Italy, Aquila
Pakistan, Khyber Pakhtunkhwa
India, —

China, Inner Mongolia
USA, the Pacific Northwest
USA, the Pacific Northwest
USA, the Pacific Northwest
USA, the Pacific Northwest
USA, California
USA, the Pacific Northwest
Mexico, Mexico State
China, Zhejiang
Mexico, Queteraro
Switzerland, Montricher
USA, the Pacific Northwest
USA, Clackamas County
Spain, —

Canada, British Columbia
Canada, British Columbia
Italy, Belluno
USA, Indiana
China, Sichuan
China, Sichuan
China, Yunnan
China, Yunnan
China, Yunnan
China, Yunnan
China, Yunnan
China, Yunnan
China, Yunnan
China, Yunnan
China, Yunnan
China, Yunnan
China, Yunnan
China, Yunnan
China, Yunnan
China, Yunnan
China, Yunnan
China, Yunnan
China, Yunnan
China, Yunnan
China, Yunnan
USA, Tennessee
USA, Wisconsin
USA, Wisconsin

GenBank Accession

No. (ITS)
MK705863*
MK705864*
MK705865*
MT447468*
MK705848*
MK705849*
MK705850*

EU526000

EU669308

EU669202

EU834202

EU846242

KU973835
MH979250
MT452507
HQ379937
MT012805
MK705866*

EU624408

EU624410

EU624411

EU834196

DQ974709

EU669206

KT874982

JQ991679
MH304404
MK028378

EU669207

JX275756

AJ292288

DQ097871

HQ650728

MT452508
MN906166
MK705867*
MK705868*
MK705869*
MK705870*
MK705871*
MK705872*
MK705873*
MK705874*
MK705875*
MK705876*
MK705877*
MK705878*
MK705879*
MK705880*
MK705881*
MK705882*
MK705883*
MK705884*
MK705885*
MK705886*
MK705887*

FJ596788

kJ140598

KJ140634